The psoralens are a group of photoreactive compounds that are activated by radiation falling within a broad range of wavelengths. Photoactivation of a psoralen molecule yields a transient species which is capable of covalently binding to a cellular target molecule in a manner which severely interrupts cellular function. The transient species remains reactive for microseconds, long enough to be chemically reactive and short enough so that, for practical purposes, once the source of irradiation is turned off, the unbound psoralen reverts to its inert form. As a consequence of these unique characteristics, psoralens provide the clinician with a class of pharmacologic agents having high chemotherapeutic potency, but only where and when the psoralens come in contact with light of an appropriate wavelength (Edelson, R., "Photopheresis: A Clinically Relevant Immunobiologic Response Modifier", Annals of N.Y. Academy of Sciences 636:154-164 (1991)).
one of the most widely used psoralens, and the only psoralen currently approved for clinical use, is 8-methoxypsoralen (8-MOP). 8-MOP is a linear furocoumarin and has an absorption spectrum which shows substantial absorption of energy at wavelengths between about 200 nm and 320 nm, a precipitous drop in absorption at wavelengths between 320 and 400 nm and very little absorption at wavelengths greater than 400 nm (visible wavelengths). In view of these well known absorption characteristics, psoralens historically have been activated by energy in the ultraviolet end of the energy spectrum.
In the presence of ultraviolet A radiation (UVA, 320 nm-400 nm), 8-MOP is capable of reacting with a wide spectrum of target molecules, e.g., cellular DNA, to form a mixture of photoadducts. Recently, Gasparro, et al., demonstrated that a form of DNA located on the cell surface, cell membrane DNA (CM-DNA), could be photomodified by 8-MOP in the presence of ultraviolet radiation (Gasparro, F., et al., "Cell membrane DNA: a new target for psoralen photoadduct formation", Photochem. Photobiol. 52:315-321 (1990). In a follow up study, Perez, et al., demonstrated that 8-MOP photomodified CM-DNA appears to play a role in immunologically mediated events (Perez, M. I., et al., "DNA associated with the cell membrane is involved in the inhibition of the skin rejection response induced by infusions of photodamaged alloreactive cells that mediate rejection of skin allograft", Photochem. Photobiol. 58:839-849 (1992).
In addition, numerous studies have reported on the effects of 8-MOP and ultraviolet A radiation on amino acids and proteins, although no 8-MOP amino acid adduct has been fully characterized (Midden, W. R., "Chemical mechanisms of the bioeffects of furocoumarins: the role of reactions with proteins, lipids, and other cellular constituents", Psoralen-DNA Photobiology (Edited by F. P. Gasparro), Vol. II:1-19, CRC Press Boca Raton, Fla. (1988) and references therein; Schlavon, O. and Veronese, F., "Extensive crosslinking between subunits of oligomeric proteins induced by furocoumarins plus UV-A irradiation," Photochem. Photobiol. 43:243-246 (1986); Granger, M. and Helene, C., "Photoaddition of 8-methoxypsoralen to E. coli DNA polymerase 1. Role of psoralen photoadducts in the photosensitized alterations of pol 1", Photochem. Photobiol. 38:563-568 (1983); Megaw, J., et al., "NMR analyses of tryptophan-8-methoxypsoralen photoreaction products formed in the presence of oxygen", Photochem. Photobiol. 32:265-269 (1990)). Midden, et al. and Caffieri, et al. have independently characterized psoralen lipid photoadducts. (Midden, W. R., Psoralen-DNA Photobiology, supra.; Specht, K. G., et al., "A new biological target of furocoumarins: photochemical formation of covalent adducts with unsaturated fatty acids," Photochem. Photobiol. 47:537-541 (1989)) (Caffieri, S., et al., "Formation of photoadducts between unsaturated fatty acids and furocoumarins", Med. Biol. Environ. 15:11-14 (1987)). However, only psoralen-DNA photoadducts have been fully characterized (Cimino, G. D., et al., "Psoralens as photoactive probes of nucleic acid structure and function: organic chemistry, photochemistry, and biochemistry", Ann. Rev. Biochem. 54:1151-1193 (1985); Kanne, D., et al., "Psoralen-deoxyribonucleic acid photoreaction. Characterization of the monoaddition products from 8-methoxypsoralen and 4,5',8-trimethylpsoralen", Biochemistry 21:861-871 (1982); Tessman, J. W., et al., "Photochemistry of the furan-side 8-methoxypsoralen-thymine monoadduct inside the DNA helix. Conversion to diadduct and to pyrone-side monoadduct", Biochemistry 24:1669-1676 (1985)).
There are three principal types of psoralen-DNA photoadducts: furan ring-DNA monoadducts (4', 5'-monoadducts or 4',5'-MA), pyrone (coumarin) ring-DNA monoadducts (3,4-monoadducts or 3,4-MA), and psoralen-DNA crosslinks. As used herein, the term monoadduct refers to a photoreaction product in which the reactive group of a target molecule, such as a pyrimidine base of DNA, is covalently coupled to either the furan ring or the pyrone ring of a psoralen molecule. The terms psoralen-crosslink or psoralen-diadduct refer to a photoreaction product in which the psoralen molecule is covalently coupled through both the furan ring and the pyrone ring to two different reactive groups of at least one target molecule, e.g., the crosslink is formed between two pyrimidine bases located on different strands of a DNA double helix.
A diverse population of psoralen-DNA photoadducts is formed as a result of the sequential absorption of photons by psoralen and psoralen-monoadducts. Upon absorbing a first photon of ultraviolet A (UVA), 8-MOP is activated to a transient species which forms a covalent bond to a reactive group of a target molecule, e.g., a pyrimidine base of a DNA, to form a psoralen-DNA monoadduct. Theoretically, either ring (furan or pyrone) may be activated with UVA radiation to form the psoralen-DNA monoadduct.
Psoralen monoadducts represent a relatively small percentage (less than 50%) of the total photoadducts formed upon activation of psoralen under photochemotherapeutic reaction conditions, such as those used in photopheresis (described below). This is because the monoadducts are capable of absorbing a second photon of radiation to form a psoralen-DNA crosslink between reactive groups of the target. In general, when the target molecule is DNA, a psoralen crosslink is formed between pyrimidine bases located on two different strands of a DNA helix. As a result of crosslink formation, DNA replication is prevented. In effect, the psoralens extend the range of wavelengths that can be used to photochemically damage DNA from wavelengths less than about 310 nm (at which wavelengths DNA is subject to direct photochemical damage from the radiation) to the long-wavelength ultraviolet (UVA wavelengths, 320-400 nm).
The synthesis and characterization of the three principal psoralen-DNA photoadducts formed between HMT 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT, a synthetic derivative of 8-MOP), and a low molecular weight synthetic oligonucleotide duplex have been described (Spielmann, H. et al., "New Methods for the Large-Scale Synthesis of Furanside Psoralen Monoadducts and Diadducts", Photochem. Photobiol. 53:1135 (1991)). Spielmann, et al. report that reaction of a DNA 12mer (a DNA oligomer containing 12 bases) and a complementary DNA 8mer (8 bases) with HMT, in the presence of krypton laser irradiation at 406.7 nm, yielded 14% 12mer furan ring monoadducts and 10% 8mer furan ring monoadducts. Irradiation of the above-described oligomers and HMT in the presence of an argon ion laser (wavelength=366 nm) yielded between 50-80% psoralen-DNA crosslinks (diadducts).
In a related work, Sastry, et al. prepared the above-described photoadducts by reacting an excess amount of psoralen with oligonucleotide duplex, i.e., 1.6.times.10.sup.-4 M HMT (48 ug/ml) to 0.4.times.10.sup.-4 M oligonucleotide duplex, in the presence of a krypton (406.7 nm or 413 nm) or argon (364 nm) laser (Sastry, S., et al., "Recent advances in the synthesis and structure determination of site specific psoralen-modified DNA oligonucleotides", J. Photochem. Photobiol. B: Biol. 14:65-79 (1992)). Sastry, et al. report that irradiation of the HMT/oligonucleotide reaction mixture at 406.7 nm yielded a mixture of furan ring-oligonucleotide monoadducts and unmodified oligonucleotides. Pyrone ring monoadducts were not detected when the krypton laser was used to synthesize monoadducts at the longer wavelength. Irradiation of the oligonucleotide duplex in the presence of HMT at 364 nm with an argon laser yielded an HMT-oligonucleotide duplex crosslink. Pyrone ring-oligonucleotide monoadducts were produced from the HMT-crosslink by reversing the furan ring-duplex bond under basic conditions.
It is not clear whether the photoadducts observed by Sastry, et al. would in fact be formed under typical photochemotherapeutic conditions, e.g., irradiation of a heterogeneous preparation of cells in the presence of a substantially lower psoralen concentration (1 ug/ml). 8-MOP is significantly less soluble than HMT and cannot be obtained in solution at the concentration used by Sastry, et al. Moreover, unlike conventional photopheresis, the Sastry, et al. photochemical reaction contained a homogeneous population of target molecules in the absence of other potentially interfering cellular components. In view of the multitude of potential reactions between psoralen and the array of molecules present in a photopheresis cell preparation ("T Cell Molecular Targets for Psoralens", Annals of N.Y. Academy of Science 636:196-208 (1991), Malane, M. and Gasparro, F.), it is unlikely that the photoadducts described by Sastry, et al. would be formed under typical photochemotherapeutic conditions.
Although long wavelength ultraviolet radiation (UVA, 320-400 nm) and 8-methoxypsoralen (8-MOP) is an established photochemotherapy for treating disorders of the immune system, the molecular basis for the therapeutic effect of psoralen photochemotherapy has not been elucidated. Formation of psoralen-DNA photoadducts (monoadducts and crosslinks) has been presumed to be responsible for the efficacy of these therapies. The therapeutic effects of psoralen photochemistry may be dependent upon the class of adduct formed as well as upon the DNA context of the adduct (Averbeck, D., "Mutagenesis by psoralens on eukaryotic cells", Photosensitization, ed. G. Moreno NATO ASI Series 15:279-291 (1988); Averbeck, D., "Recent advances in psoralen phototoxicity mechanism", Photochem. Photobiol. 50:859-882 (1989) Sage, E., and Bredberg, A., "Damage distribution and mutation spectrum: the case of 8-methoxypsoralen plus UVA in mammalian cells", Mutation Research 263:217-222 (1991)); Cundari, E., Averbeck, D., "8-Methoxypsoralen-photoinduced DNA crosslinks as determined in yeast by alkaline step elution under different reirradiation conditions. Relation with genetic effects", Photochem. Photobiol. 48:315-320 (1988); Bredberg, A. and Nachmansson, N., "Psoralen adducts in a shuttle vector plasmid propagated in primate cells: High mutagenicity of DNA cross-links," Carcinogenesis 8:1923-1927 (1987); Averbeck, D., et al., "Mutagenic and recombinogenic action of DNA monoadducts photoinduced by the bifunctional furocoumarin 8-Methoxypsoralen in yeast (Saccharomyces cerevisiae)", Photochem. Photobiol. 45:371-379 (1987); Cassier, C., et al., "Mutagenic and recombinogenic effects of DNA cross-links induced in yeast by 8-methoxypsoralen photoaddition", Photochem. Photobiol. 39:799-803 (1984)), Shinghal, R. P., et al., "High-performance liquid chromatography for trace analysis of DNA and kinetics of CNA modification", BioChrom. 4:78-88 (1989); Sage, E., and Moustacchi, E., "Sequence context effects on 8-methoxypsoralen photobinding to defined DNA fragments", Biochemistry 26:3307-3314 (1987)).
The ability of a cell line to repair lesions also affects the toxicity and mutagenicity of psoralen-DNA photoadducts (Potapenko, A. Y., "Mechanisms of photodynamic effects of furocoumarins", J. Photochem. Photobiol. B. 9:1-33 (1991); Babudri, N., et al., "Mutation Induction and killing of V79 chinese hamster cells by 8-methoxypsoralen plus near-ultraviolet light: relative effects of monoadducts and crosslinks", Mutation Res. 91:391-394 (1981)). Accordingly, cellular mutations may arise as a consequence of misrepair or misreading during mitosis of the psoralen-DNA lesion. Indirect evidence suggests that the psoralen-DNA crosslink may be the most mutagenic of the psoralen-DNA lesions (Averbeck, D., Photosensitization, NATO ASI Series 15:279-291 (1988) supra; Cundari, E., Averbeck, D., Photochem. Photobiol. 48:315-320 (1988) supra; Bredberg, A. and Nachmansson, N., Carcinogenesis 8:1923-1927 (1987) supra; Sage, E., Bredberg, A., Mutation Research 263:217-222 (1991) supra; Cassier, C., et al., Photochem. Photobiol. 39:799-803 (1984) supra).
Psoralen photochemotherapy has been shown to alter cellular antigenicity (Malane, M., et al., "Treatment with 8-methoxypsoralen (8-MOP) and ultraviolet A (UVA) radiation enhances immune recognition in a murine mastocytoma model", J. Invest. Dermatol. 98:554A (1992)). It is believed that psoralen photoadduct formation modulates the immune system response by causing cellular mutations which result in phenotypic cellular changes. Accordingly, psoralen photochemotherapy has been directed to (1) regulating an aberrant immune system response via modification of the T cell surface receptor, e.g., by inducing presentment of a novel T cell receptor or (2) augmenting the immune system response to a specific antigen, e.g., by inducing presentment of novel antigenic peptides associated with major histocompatibility complex molecules on the cell surface. Several studies, described herein, have suggested that the ability of the immune system to recognize the receptor of an aberrant T cell clone as antigenic makes possible the vaccination of a patient against a pathogenic clone of T cells, i.e., by administering to the patient photochemically modified aberrant T cells.
Cutaneous T cell lymphoma (CTCL) is an example of an immune system disease that is caused by a massive expansion of a single clone of aberrant T cells. Extracorporeal photochemotherapy ("photopheresis") for the treatment of cutaneous T cell lymphoma has been described (Edelson, R., "Light-activated Drugs", Scientific American 256(8): 68-75 (1988); Edelson, R., Annals of N.Y. Academy of Sciences 636:154-164 (1991) supra. The treatment comprises isolating the patient's T cells, irradiating the cells with ultraviolet A radiation in the presence of a photoactivatable agent, e.g., 8-MOP, and reinfusing the damaged T cells. This therapy reportedly results in selective destruction of the malignant T cell clone.
Conventional photopheresis (irradiation with ultraviolet A radiation in the presence of 8-MOP) has also been used for the treatment of several autoimmune disorders, including pemphigus vulgaris and systemic sclerosis (Rook, A., "Photopheresis in the Treatment of Autoimmune Disease: Experience with Pemphigus Vulgaris and Systemic Sclerosis", Annals of N.Y. Academy of Science 636:209-216 (1991) and rheumatoid arthritis (Malawista, S., et al., "Photopheresis for Rheumatoid Arthritis", Annals of N.Y. Academy of Science 636:217-226 (1991). Long wavelength ultraviolet radiation and 8-MOP have also been used as a photochemotherapy for the treatment of psoriasis (Parrish, J., et al., "Photochemotherapy of psoriasis with oral methoxsalen and long wavelength ultraviolet light", N. Engl. J. Med. 29:1207-1211 (1974)) and in a preliminary study to evaluate the potential therapeutic value of photopheresis in seven patients with AIDS-related complex (ARC) (Bisaccia, E. et al., "Viral-Specific Immunization in AIDS-Related Complex by Photopheresis", Annals of N.Y. Academy of Science 636:321-330 (1991).
Photopheresis also has been used prophylactically to prevent graft rejection by injecting into mice a preparation containing Photoinactivated Effector T ("PET") cells (Perez, M. et al., "Inhibition of Antiskin Allograft Immunity Induced by Infusions with Photoinactivated Effector T Lymphocytes (PET Cells); "The Congenic Model", Transplantation 51:1283-1289 (1991). To prepare the PET cells, T cell clones mediating skin graft rejection were expanded in vivo and photoinactivated by ultraviolet irradiation in the presence of 8-MOP. Perez, et al. report that this procedure results in the adoptive transfer of tolerance for skin allotransplantation, as demonstrated by prolongation of allograft survival in the recipients of PET cells.
The above-described photopheresis procedures have also been used to augment the immune system response to a specific antigen. In particular, U.S. Pat. No. 4,838,852, issued to Edelson et al. (hereinafter Edelson '852), the contents of which are incorporated herein by reference, describes a method for enhancing the immune system response of a mammal to an antigen. The Edelson '852 method comprises (a) contacting the subject's immune system with the specific antigen for a suitable time to artificially stimulate the immune system, (b) withdrawing antigen-stimulated blood cell material from the subject, (c) treating the withdrawn material to alter the antigen-stimulated cells, and (d) returning the treated material to the subject. Edelson '852 also discloses that it may be possible to render the cells incapable of recognizing an antigen by withdrawing the blood cell-containing material from the subject, treating the withdrawn material as above, returning the treated material to the subject and then contacting the subject's immune system with a specific antigen.
U.S. Pat. No. 5,147,289, issued to Edelson (hereinafter Edelson '289), the contents of which are incorporated herein by reference, describes methods for non-specifically enhancing the immune system response of a mammal to an antigen. The method comprises (A) enhancing the immune system response by (a) withdrawing leukocyte containing material from the mammal, (b) treating the withdrawn leukocytes in a manner to alter the cells, (c) returning the treated leukocytes to the mammal and (B) artificially contacting the mammal's immune system with the antigen for a suitable period of time to stimulate an immune system response.
With respect to the Edelson '852 and '289 patents, the withdrawn leukocytes may be altered by, for example, irradiating the leukocytes with ultraviolet radiation in the presence of a photoactivatable agent, e.g., a psoralen, exposure to ultraviolet light in the absence of a photoactivatable agent, and exposure to visible light in the presence of a photoactivatable agent such as hematoporphyrin. In a preferred embodiment, the leukocytes are gently damaged by ultraviolet radiation in the presence of a psoralen.
The combination of ultraviolet radiation (320-400 nm) and 8-MOP has also been proposed as a photochemotherapeutic method for preventing restenosis in a subject undergoing vascular recanalization (U.S. Pat. No. 5,116,864, issued to March et al.). Ultraviolet radiation in the range of about 320 to about 400 nm was disclosed to activate the psoralen at the region of the recanalization. More recently, L. Deckelbaum, et al. described a method for preventing restenosis, which method includes activation of 8-MOP with visible light irradiation to inhibit smooth muscle cell proliferation (Deckelbaum, L. et al., "Inhibition of Smooth Muscle Cell Proliferation by 8-Methoxypsoralen Photoactivated by Visible Light", American Heart Assoc. 65th Scientific Session, abstract no. 135231 (1992)). Bovine aortic smooth muscle cells were exposed to 1 ug/ml 8-MOP and irradiated with a broadband blue light source (peak wavelength=420 nm, bandwidth=34 nm). The formation of 8-MOP-DNA adducts in smooth muscle cells was confirmed by HPLC analysis.
None of the above-cited references and patents disclose a method for modulating an immune system response, which method comprises administering a cell suspension containing a plurality of non-lethal psoralen-photoadducts (monoadducts) and substantially no lethal psoralen-photoadducts (crosslinks). Accordingly, there is still a need for psoralen photoactivation methods which permit formation of photochemically modified cells containing psoralen-DNA monoadducts under conditions which generate virtually no psoralen-DNA crosslinks. The photoactivation method would be less toxic to the irradiated cells in comparison with photopheresis conditions currently employed.